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Direct repeats of T‐DNA integrated in tobacco chromosome: characterization of junction regions
Author(s) -
Krizkova Lucie,
Hrouda Milan
Publication year - 1998
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1998.00330.x
Subject(s) - dna , biology , direct repeat , plasmid , transformation (genetics) , agrobacterium , genetics , inverted repeat , chromosome , genome , microbiology and biotechnology , gene
Summary Plant transformation viaAgrobacteriumfrequently results in formation of multiple copy T‐DNA arrays at one target site of the chromosome. The T‐DNA copies are arranged in repeats, direct or inverted around one of the T‐DNA borders. A Ti plasmid‐derived transformation vector has been constructed enabling direct selection of transformants carrying at least two linked copies of T‐DNA in the same orientation. The selection is based on expression of a promoterless neomycin phosphotransferase gene on one T‐DNA copy from a promoter located on the other T‐DNA copy. After co‐cultivation of tobacco protoplasts withAgrobacterium, as many as 30% of regenerated transformed plants carried directly repeated T‐DNA copies. The junction regions between two T‐DNAs were amplified and 13 amplified fragments were cloned and sequenced. The involvement of T‐DNA left and right border sequences in direct repeat junctions was determined. In some junctions, additional filler DNA was detected. The length of filler DNA varied from a few up to almost 300 bp. The longer filler DNAs from two clones were found to be T‐DNA fragments in direct or reverse orientation. We discuss the recently suggested models for T‐DNA integration and propose that the formation of direct repeats in genomes does not necessarily result from ligation of intermediates (i.e. T‐strands), but more likely from the co‐integration of several intermediates into one target site.